In order to solve recent problems of exhaustion and price increase of fossil resources and global warming, a process for producing chemical products without dependence on fossil resources has been desired. As a solution to such problems, biorefinery technology utilizing biomass resources, in particular inedible biomass resources as raw materials, has attracted keen attention.
Aniline is widely used as a starting raw material for the synthesis of dyes; functional polymers; chemical agents for tire rubber, including a vulcanization accelerator and an antioxidant; agricultural chemicals; medicinal drugs; or the like.
Currently, aniline is produced by chemical conversion from crude oil as a raw material. Specifically, benzene obtained from naphtha obtainable by distillation separation of crude oil is used as the raw material. More specifically, a process in which nitrobenzen obtained by nitration of benzene and a metal catalyst, such as copper or nickel, are used; a process in which nitrobenzen is hydrogenated by reduction using iron and hydrochloric acid; or a process in which chlorobenzene and ammonia are reacted at high temperature and pressure may be used.
However, these methods depend on fossil materials for benzene as the starting material, and in addition, need high-temperature and high-pressure conditions in the reaction process, causing problems of consumption of an enormous amount of fossil fuel and emissions of greenhouse gases, such as carbon dioxide. For the reason, the development of an environment-conscious bioprocess which uses renewable resources as raw materials and does not depend on fossil resources for the raw materials in the production process has been desired.
For bioprocess production of an aromatic compound, such as aniline, a synthetic pathway called shikimate pathway possessed by microorganisms and plants can be used. However, such a process, which consists of a great many reaction steps for the metabolism of raw material sugars, is less productive.
Further, when aniline is produced by a bioprocess, due to the cytotoxicity, the bacterial growth inhibitory effect, etc. of the produced aniline, the bacterial growth per se is inhibited. Therefore, industrial production of aniline by a bioprocess is very difficult.
Aniline production technologies using non-genetically-modified bacteria that have been disclosed so far include an example where aniline was produced by adding 4-aminobenzoic acid to Mycobacterium smegmatis (Non Patent Literature 1), and an example where aniline was produced by adding anthranilic acid or 4-aminobenzoic acid to virulent Escherichia coli O111:B4 cells or extracts thereof (Non Patent Literature 2).
However, in these documents, no enzyme molecule or enzyme gene involved in the aniline production was specified, and no later reports have been made. Non Patent Literature 1 reported aniline production from 7.3 mM 4-aminobenzoic acid, but whether the substance identification was correct or not is uncertain because the produced aniline was only a slight amount (6.9 μM in concentration) and the identification was performed based on comparison using paper chromatography.
The process of Non Patent Literature 2 is not suitable for industrial production because the microorganism used therein is virulent. In addition, the aniline identification is indirectly performed based on diazo coloring, and whether the substance identification was correct or not is also uncertain. Further, aniline generally inhibits the growth of the microorganism due to its cytotoxicity, and therefore production of high levels of aniline is rather difficult.
The present inventors have already found that a transformant constructed by introducing a gene which is derived from Enterobacter cloacae and which encodes an enzyme having aminobenzoate decarboxylase activity into a coryneform bacterium as a host produces 1.25 mM aniline from 5 mM 4-aminobenzoic acid (Patent Literature 1). However, for cost reduction in industrial production, the production of higher levels of aniline is required, and therefore the aminobenzoate decarboxylase activity needs to be further improved.